Ximing Guo

The oyster genome reveals stress adaptation and complexity of shell formation

The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster’s adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.

Oyster Reefs at Risk and Recommendations for Conservation, Restoration, and Management

Native oyster reefs once dominated many estuaries, ecologically and economically. Centuries of resource extraction exacerbated by coastal degradation have pushed oyster reefs to the brink of functional extinction worldwide. We examined the condition of oyster reefs across 144 bays and 44 ecoregions; our comparisons of past with present abundances indicate that more than 90% of them have been lost in bays (70%) and ecoregions (63%). In many bays, more than 99% of oyster reefs have been lost and are functionally extinct. Overall, we estimate that 85% of oyster reefs have been lost globally. Most of the worlds remaining wild capture of native oysters (> 75%) comes from just five ecoregions in North America, yet the condition of reefs in these ecoregions is poor at best, except in the Gulf of Mexico. We identify many cost-effective solutions for conservation, restoration, and the management of fisheries and nonnative species that could reverse these oyster losses and restore reef ecosystem services.

All-triploid Pacific oysters (Crassostrea gigas Thunberg) produced by mating tetraploids and diploids

Abstract To document the reproductive characteristics of tetraploids and determine whether they can be used for triploid production, factorial crosses were made between diploids (D) and tetraploids (T), producing DD, DT, TD and TT groups (female listed first). A normal triploid group was also produced by blocking polar body II with cytochalasin B (3nCB). Survival to spat in TD and DT groups was about the same as in normal diploids, and significantly higher than in the 3nCB and TT groups. As determined by flow cytometry, all surviving oysters from DT and TD crosses were triploids, and only 46% of oysters from the 3nCB group were triploids. TT crosses produced primarily tetraploids despite low survival. At 8 and 10 months of age, triploid oysters from DT and TD groups were 13–51% larger than normal diploids, possibly due to polyploid gigantism. These results suggest that mating tetraploids and diploids is the best method for triploid production, and triploids produced in this way are better suited for aquaculture than those produced by altering meiosis and are ideal for population control.

AFLP-based genetic linkage maps of the Pacific oyster Crassostrea gigas Thunberg

Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.

Genetic linkage map of the eastern oyster Crassostrea virginica Gmelin

Amplified fragment length polymorphisms (AFLPs), along with some microsatellite and Type I markers, were used for linkage analysis in Crassostrea virginica Gmelin, the eastern oyster. Seventeen AFLP primer combinations were selected for linkage analysis with two parents and their 81 progeny. The 17 primer combinations produced 396 polymorphic markers, and 282 of them were segregating in the two parents. Chi-square analysis indicated that 259 (91.8%) markers segregated in Mendelian ratio, while the other 23 (8.2%) showed significant (P < 0.05) segregation distortion, primarily for homozygote deficiency and probably due to deleterious recessive genes. Moderately dense linkage maps were constructed using 158 and 133 segregating markers (including a few microsatellite and Type I markers) from male and female parents, respectively. The male framework map consisted of 114 markers in 12 linkage groups, covering 647 cM. The female map had 84 markers in 12 linkage groups with a length of 904 cM. The estimated genome length was 858 cM for the male map and 1296 cM for the female map. The observed genome coverage was 84% for the male and female map when all linked markers were considered. Genetic maps observed in this study are longer than the cytogenetic map, possibly because of low marker density.

Construction of Genetic Linkage Maps and Comparative Genome Analysis of Catfish Using Gene-associated Markers

A genetic linkage map of the channel catfish genome (N = 29) was constructed using EST-based microsatellite and single nucleotide polymorphism (SNP) markers in an interspecific reference family. A total of 413 microsatellites and 125 SNP markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 allowed mapping of 331 markers (259 microsatellites and 72 SNPs) to 29 linkage groups. Each linkage group contained 3–18 markers. The largest linkage group contained 18 markers and spanned 131.2 cM, while the smallest linkage group contained 14 markers and spanned only 7.9 cM. The linkage map covered a genetic distance of 1811 cM with an average marker interval of 6.0 cM. Sex-specific maps were also constructed; the recombination rate for females was 1.6 times higher than that for males. Putative conserved syntenies between catfish and zebrafish, medaka, and Tetraodon were established, but the overall levels of genome rearrangements were high among the teleost genomes. This study represents a first-generation linkage map constructed by using EST-derived microsatellites and SNPs, laying a framework for large-scale comparative genome analysis in catfish. The conserved syntenies identified here between the catfish and the three model fish species should facilitate structural genome analysis and evolutionary studies, but more importantly should facilitate functional inference of catfish genes. Given that determination of gene functions is difficult in nonmodel species such as catfish, functional genome analysis will have to rely heavily on the establishment of orthologies from model species.

Massive expansion and functional divergence of innate immune genes in a protostome.

The molecules that mediate innate immunity are encoded by relatively few genes and exhibit broad specificity. Detailed annotation of the Pacific oyster (Crassostrea gigas) genome, a protostome invertebrate, reveals large-scale duplication and divergence of multigene families encoding molecules that effect innate immunity. Transcriptome analyses indicate dynamic and orchestrated specific expression of numerous innate immune genes in response to experimental challenge with pathogens, including bacteria, and a pathogenic virus. Variable expression of individual members of the multigene families encoding these genes also occurs during different types of abiotic stress (environmentally-equivalent conditions of temperature, salinity and desiccation). Multiple families of immune genes are responsive in concert to certain biotic and abiotic challenges. Individual members of expanded families of immune genes are differentially expressed under both biotic challenge and abiotic stress conditions. Members of the same families of innate immune molecules also are transcribed in developmental stage- and tissue-specific manners. An integrated, highly complex innate immune system that exhibits remarkable discriminatory properties and responses to different pathogens as well as environmental stress has arisen through the adaptive recruitment of tandem duplicated genes. The co-adaptive evolution of stress and innate immune responses appears to have an ancient origin in phylogeny.

Reproductive potential and genetics of triploid Pacific oysters, Crassostrea gigas (Thunberg)

The reproductive potential and genetics of triploidy were studied in the Pacific oyster. DNA content in sperm from triploids showed a single peak at 1.5c as determined by flow cytometry. In eggs from triploids, trivalents were the dominant form of synapsed chromosomes, although the degree of synapsis varied considerably within and among females. Some eggs went through complete synapsis and formed 10 trivalents, chromosomes; most had a mixture of 11-13 trivalents, bivalents, and univalents. Factorial matings were produced from diploid (D) and triploid (T) parent oysters, creating four crosses: DD, DT, TD, and TT (female first). Gametes from triploids were fully capable of fertilization. After fertilization, eggs from triploids went through two meioses and released two polar bodies as diploid eggs did. Karyological analyses showed that average ploidy of the resultant embryos was 2.0 n for DD, 2.46 n for DT, 2.52 n for TD, and 2.88 n for TT. Survival of fertilized eggs to metamorphosis and settlement was about 21% for DD, but considerably lower on other crosses: 0.0007% for DT, 0.0463% for TD, and 0.0085% for TT. Nine months after matings, all survivors from DT crosses were diploid. Survivors from TD crosses consisted of 33% diploids, 57% triploids, and 10% tetraploids. Survivors from the TT crosses consisted of 90% triploids, 4% diploids, and 6% mosaics. We hypothesize that differences in ploidy composition between DT and TD embryos and survivors were caused by pro-egg segregations that favor the retention, rather than loss, of extra chromosomes in the egg. The reproductive potential of triploids and evolutionary implications are discussed.

GENETIC DETERMINANTS OF PROTANDRIC SEX IN THE PACIFIC OYSTER, CRASSOSTREA GIGAS THUNBERG

A unique feature of sex in Crassostrea oysters is the coexistence of protandric sex change, dioecy, and hermaphroditism. To determine whether such a system is genetically controlled, we analyzed sex ratios in 86 pair‐mated families of the Pacific oyster, Crassostrea gigas Thunberg. The overall female ratios of one‐, two‐, and three‐year‐old oysters were 37%, 55%, and 75%, respectively, suggesting that a significant proportion of oysters matured first as males and changed to females in later years. Detailed analysis of sex ratios in factorial and nested crosses revealed significant paternal effects, which corresponded to two types of sires. No major maternal effects on sex were observed. Major genetic control of sex was further indicated by the distribution of family sex ratios in two to four apparently discreet groups. These and other data from the literature are compatible with a single‐locus model of primary sex determination with a dominant male allele (M) and a protandric female allele (F), so that MF are true males and FF are protandric females that are capable of sex change. The rate of sex change of FF individuals may be influenced by secondary genes and/or environmental factors. Strong maternal and weak paternal effects on sexual maturation or time of spawning were also suggested.

THE CASE FOR SEQUENCING THE PACIFIC OYSTER GENOME

Abstract An international community of biologists presents the Pacific oyster Crassostrea gigas as a candidate for genome sequencing. This oyster has global distribution and for the past several years the highest annual production of any freshwater or marine organism (4.2 million metric tons, worth